Improvement in survival and cardiac metabolism after gene transfer of sarcoplasmic reticulum Ca(2+)-ATPase in a rat model of heart failure.

BACKGROUND: In heart failure, sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) activity is decreased, resulting in abnormal calcium handling and contractile dysfunction. We have previously shown that increasing SERCA2a expression by gene transfer improves ventricular function in a rat model of heart failure created by ascending aortic constriction. METHODS AND RESULTS: In this study, we tested the effects of gene transfer of SERCA2a on survival, left ventricular (LV) volumes, and metabolism. By 26 to 27 weeks after aortic banding, all animals developed heart failure (as documented by >25% decrease in fractional shortening) and were randomized to receive either an adenovirus carrying the SERCA2a gene (Ad.SERCA2a) or control virus (Ad.betagal-GFP) by use of a catheter-based technique. Sham-operated rats, uninfected or infected with either Ad.betagal-GFP or Ad.SERCA2a, served as controls. Four weeks after gene transfer, survival in rats with heart failure treated with Ad.betagal-GFP was 9%, compared with 63% in rats receiving Ad.SERCA2a. LV volumes were significantly increased in heart failure (0.64+/-0.05 versus 0.35+/-0.03 mL, P<0.02). Overexpression of SERCA2a normalized LV volumes (0.46+/-0.07 mL) in the failing hearts. (31)P NMR analysis showed a reduced ratio of phosphocreatine to ATP content in failing+Ad.betagal-GFP compared with sham+Ad.betagal-GFP (0.82+/-0.13 versus 1.38+/-0.14, P<0.01). Overexpression of SERCA2a in failing hearts improved the phosphocreatine/ATP ratio (1.23+/-0.28). CONCLUSIONS: In this study, we show that unlike inotropic agents that improve contractile function at the expense of increased mortality and worsening metabolism, gene transfer of SERCA2a improves survival and the energy potential in failing hearts.

Myofilament calcium regulation in human myocardium.

BACKGROUND: We investigated whether decreased myofilament calcium contractile activation may, in part, contribute to heart failure. METHODS AND RESULTS: Calcium concentration required for 50% activation and Hill coefficient for fibers from nonfailing and failing human hearts at pH 7.1 were not different. Maximum calcium-activated force (F(max)) was also not different. However, at pH 6.8 and 6.9, differences were seen in myofilament calcium activation between nonfailing and failing hearts. At lower pH, failing myocardium was shifted left on the calcium axis compared with nonfailing myocardium, which suggested an increase in myofilament calcium responsiveness. Increased inorganic phosphate concentration decreased maximal force development by 56% in nonfailing and 36% in failing myocardium and shifted the calcium-force relationship by 2.01+/-0.22 versus 0.86+/-0.13 micromol/L, respectively (P<0.05). Addition of cAMP resulted in a 0. 56 micromol/L shift toward higher intracellular calcium concentrations in nonfailing myocardium and a 1.04 micromol/L shift in failing myocardium. Protein kinase A in the presence of cAMP resulted in a further rightward shift in nonfailing human myocardium but did not further shift the calcium-force relationship in fibers from failing hearts. cGMP also resulted in a greater decrease in myofilament calcium sensitivity in fibers from failing hearts. CONCLUSIONS: We propose that changes at the level of the thin myofilaments result in differential responses to changes in the intracellular milieu in nonfailing versus failing myocardium.

Human heart failure: cAMP stimulation of SR Ca(2+)-ATPase activity and phosphorylation level of phospholamban.

Failing human myocardium has been associated with decreased sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity. There remains controversy as to whether the regulation of SR Ca(2+)-ATPase activity is altered in heart failure or whether decreased SR Ca(2+)-ATPase activity is due to changes in SR Ca(2+)-ATPase or phospholamban expression. We therefore investigated whether alterations in cAMP-dependent phosphorylation of phospholamban may be responsible for the reduced SR Ca(2+)-ATPase activity in human heart failure. Protein levels of phospholamban and SR Ca(2+)-ATPase, detected by Western blot, were unchanged in failing compared with nonfailing human myocardium. There was decreased responsiveness to the direct activation of the SR Ca(2+)-ATPase activity by either cAMP (0.01-100 micromol/l) or protein kinase A (1-30 microgram) in failing myocardium. Using the backphosphorylation technique, we observed a decrease of the cAMP-dependent phosphorylation level of phospholamban by 20 +/- 2%. It is concluded that the impaired SR function in human end-stage heart failure may be due, in part, to a reduced cAMP-dependent phosphorylation of phospholamban.

Fibroblast growth factor interactions in the developing lung.

Cellular activities that lead to organogenesis are mediated by epithelial-mesenchymal interactions, which ultimately result from local activation of complex gene networks. Fibroblast growth factor (FGF) signaling is an essential component of the regulatory network present in the embryonic lung, controlling proliferation, differentiation and pattern formation. However, little is known about how FGFs interact with other signaling molecules in these processes. By using cell and organ culture systems, we provide evidence that FGFs, Sonic hedgehog (Shh), bone morphogenetic protein 4 (BMP-4), and TGFbeta-1 form a regulatory circuit that is likely relevant for lung development in vivo. Our data show that FGF-10 and FGF-7, important for patterning and growth of the lung bud, are differentially regulated by FGF-1, -2 and Shh. In addition, we show that FGFs regulate expression of Shh, BMP-4 and other FGF family members. Our data support a model in which Shh, TGFbeta-1 and BMP-4 counteract the bud promoting effects of FGF-10, and where FGF levels are maintained throughout lung development by other FGFs and Shh.

FGF-10 is a chemotactic factor for distal epithelial buds during lung development.

Fibroblast growth factor (FGF) signaling is required for normal epithelial branching in the respiratory system of several species. Recent studies have shown that FGF-10 may be a key regulator of lung branching morphogenesis, based on its pattern of expression in the early lung and its ability to induce epithelial budding in vitro. In this study we investigate whether FGF-10 is able to direct lung epithelial buds to proper positions during development . We maintained localized high levels of FGF-10 in cultured lungs using FGF-10-soaked heparin beads. FGF-10 exerts a powerful chemoattractant effect on the distal but not on proximal lung epithelium. Epithelial buds grow toward an FGF-10 source within 24 h, and subsequently form concentric layers of epithelium around the bead. BrdU incorporation analysis suggests that FGF-10, in contrast to FGF-7, is a modest proliferation factor for the lung epithelium. In the absence of mesenchyme FGF-10 requires an associated proliferative signal to induce bud migration. This can be provided by extract from lung mesenchyme, or by FGF-7, a growth factor also present in the early embryonic lung. FGF-10 does not seem to interfere with early epithelial cell differentiation. The chemoattractant effect of FGF-10 in the lung epithelium is reminiscent of the patterning effect of the Drosophila FGF ortholog branchless in the developing tracheal epithelium, suggesting that the function of these genes has been conserved during evolution.

ERcalcistorin/protein-disulfide isomerase acts as a calcium storage protein in the endoplasmic reticulum of a living cell. Comparison with calreticulin and calsequestrin.

ERcalcistorin/protein-disulfide isomerase (ECaSt/PDI), a high capacity low affinity Ca2+-binding protein in the endoplasmic reticulum of sea urchin eggs (Lebeche, D., and Kaminer, B. (1992) Biochem. J. 287, 741-747), shares 55% sequence identity with mammalian PDI and has PDI activity (Lucero, H., Lebeche, D., and Kaminer, B. (1994) J. Biol. Chem. 269, 23112-23119). We report on ECaSt/PDI functioning as a Ca2+ storage protein in the endoplasmic reticulum (ER) of a living cell and compare it with calsequestrin and calreticulin, high capacity low affinity Ca2+-binding proteins in the sarcoplasmic reticulum and ER, respectively. Stably transfected Chinese hamster ovary cell clones expressed these proteins, which were localized in the ER of the cell. Microsomes from cells expressing ECaSt/PDI, calreticulin, and calsequestrin accumulated 17.2 +/- 0.27, 20.0 +/- 0.82, and 38.0 +/- 0.28 nmol of Ca2+/mg of protein, respectively; control microsomes accumulated from 2.6 +/- 0.17 to 2.9 +/- 0.14 nmol of Ca2+/mg of protein. The initial rate of Ca2+ uptake was similar in microsomes from transfected and control cells. Microsomes containing an ECaSt/PDI mutant in which 45% of the acidic residue pairs in the C terminus were truncated had a reduced Ca2+ storage capacity. This supports our previous hypothesis that the degree of low affinity Ca2+ binding is dependent on the number of pairs of carboxyl groups in the molecule. The maximal Ca2+ accumulation by microsomes containing the expressed ECaSt/PDI, C-terminally truncated ECaSt/PDI, calreticulin, or calsequestrin correlates approximately with the Ca2+ binding capacity of the respective proteins.

ERcalcistorin/protein disulfide isomerase (PDI). Sequence determination and expression of a cDNA clone encoding a calcium storage protein with PDI activity from endoplasmic reticulum of the sea urchin egg.

Following the purification of a 58-kDa calsequestrin-like protein from the endoplasmic reticulum (ER) of sea urchin eggs (Oberdorf, J.A., Lebeche, D., Head, J. F., and Kaminer, B. (1988) J. Biol. Chem. 263, 6806-6809) and its characterization as a high capacity, low affinity calcium-binding protein (Lebeche, D., and Kaminer, B. (1992) Biochem. J. 287, 741-747) we isolated and sequenced a cDNA encoding for this protein. The deduced 496 amino acids contain a 17-residue NH2-terminal signal peptide, a KDEL COOH-terminal ER retention signal and two thioredoxin-like active site domains, -CGHC-, identical with those in protein disulfide isomerase (PDI). The sea urchin egg protein shares a 55% sequence identity with mammalian PDI and its PDI activity is 30% of the activity of rabbit liver PDI. The corresponding mRNA was found in oocytes, mature eggs, embryos, and differentiated tissues of the sea urchin in varying amounts. COS-7 cells transfected with the cDNA, expressed a 58-kDa protein immunoreactive to antibodies against the sea urchin egg protein. This molecule appears to have a dual function of calcium storage and PDI activity within the ER. We hence redesignate it ERcalcistorin/PDI (ECaSt/PDI), a protein that is distinct from calsequestrin and calreticulin.

Calcium binding properties of rabbit liver protein disulfide isomerase.

We have characterized the Ca2+ binding properties of protein disulfide isomerase (PDI). It binds 19 mol Ca2+/mol protein with low affinity, which is reduced by increasing the ionic strength. Ca2+ induced conformational changes detected by UV difference spectroscopy. These Ca2+ binding properties resemble those of a sea urchin egg 58 kDa protein.

Characterization of a calsequestrin-like protein from sea-urchin eggs.

Following our studies on the identification of a calsequestrin-like protein (CSLP) from sea-urchin eggs [Oberdorf, Lebeche, Head & Kaminer (1988) J. Biol Chem. 263, 6806-6809], we have characterized its Ca(2+)-binding properties and identified it as a glycoprotein. The molecule binds 23 mol of Ca2+/mol of protein, as determined by equilibrium dialysis. This is in the range reported for cardiac calsequestrin but is about half the binding capacity of striated muscle calsequestrin. The affinities of the CSLP for Ca2+ are decreased by increasing KCl concentrations (20-250 mM) and the presence of Mg2+ (3 mM) in the medium: the half-maximal binding values varied from 1.62 to 5.77 mM. Hill coefficients indicated mild co-operativity in the Ca2+ binding. Ca2+ (1-8 mM)-induced u.v. difference spectra and intrinsic fluorescence changes suggest a net exposure of aromatic residues to an aqueous environment. C.d. measurements showed minor Ca(2+)-induced changes in alpha-helical and beta-sheet content of less than 10%. These spectral changes are distinctly different from those found in muscle calsequestrin. Immunoblotting studies showed that the CSLP is distinct from calreticulin, a low-affinity Ca(2+)-binding protein.

A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo.

Using an antiserum produced against a purified calsequestrin-like (CSL) protein from a microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on sea urchin eggs and embryos in the first cell cycle. The sea urchin CSL protein has been found to bind Ca++ similarly to calsequestrin, the well-characterized Ca++ storage protein in the sarcoplasmic reticulum of muscle cells. In semi-thin frozen sections of unfertilized eggs, immunofluorescent staining revealed a tubuloreticular network throughout the cytoplasm. Staining of isolated egg cortices with the CSL protein antiserum showed the presence of a submembranous polygonal, tubular network similar to ER network patterns seen in other cells and in egg cortices treated with the membrane staining dye DiIC16[3]. In frozen sections of embryos during interphase of the first cell cycle, a cytoplasmic network similar to that of the unfertilized egg was present. During mitosis, we observed a dramatic concentration of the antibody staining within the asters of the mitotic apparatus where ER is known to aggregate. Electron microscopic localization on unfertilized eggs using peroxidase-labeled secondary antibody demonstrated the presence of the CSL protein within the luminal compartment of ER-like tubules. Finally, in frozen sections of centrifugally stratified eggs, the immunofluorescent staining concentrated in the clear zone: a layer highly enriched in ER and thought to be the site of calcium release upon fertilization. This localization of a CSL protein within the ER of the egg provides evidence for the ability of this organelle to serve a Ca++ storage role in the regulation of intracellular Ca++ in nonmuscle cells in general, and in the regulation of fertilization and cell division in sea urchin eggs in particular.

Identification of a calsequestrin-like protein from sea urchin eggs.

Following studies on calcium transport by isolated smooth endoplasmic reticulum from unfertilized sea urchin eggs (Oberdorf, J. A., Head, J. F., and Kaminer, B. (1986) J. Cell Biol. 102, 2205-2210) we have purified and partially characterized a calsequestrin-like protein from this organelle isolated from eggs from Strongylocentrotus droebachiensis and Arbacia punctulata. Muscle calsequestrin from sarcoplasmic reticulum is well characterized as a calcium storage protein. The egg protein resembles calsequestrin in its behavior in purification steps, electrophoretic mobility, blue staining with Stains-all on polyacrylamide gels, and its calcium binding and amino acid composition. Purification was attained with DEAE-cellulose and hydroxyapatite chromatography. The egg protein Mr of 58,000 in the Laemmli gel system is reduced to 54,000 under Weber-Osborn (neutral) conditions, thus showing a pH dependence in its mobility, although less than occurs with muscle calsequestrins. 25% of its amino acids are acidic and 10% basic. It binds 309 nmol of Ca2+/mg of protein, within the range reported for cardiac calsequestrin. Antigenically, the sea urchin egg protein is related to cardiac calsequestrin capable of binding anti-cardiac calsequestrin antibody.